Since its discovery in 2004, livestock-associated MRSA ST398 has emerged as an increasingly common cause of human infections in Northern Europe and a prevalent colonizer of livestock and farm workers in the U.S. and around the world. A recent survey of Iowa hog farms showed that 70% of farms-and nearly half of the hog farmers-were positive for MRSA ST398. Meanwhile, the epidemiology of ST398 in Northern Europe appears to be shifting. Initially, all MRSA ST398 infections could be traced back to direct or indirect contact with livestock, but new cases without known livestock exposure are increasing, particularly in Denmark and the Netherlands. Yet, despite serious public health concerns, little is known about this rapidly emerging antibiotic-resistant pathogen. This is attributed in large part to the limitations of standard molecular analyses for elucidating the origins, evolution, and dispersal of the highly clonal ST398 lineage. To develop infection control and surveillance strategies, we must better understand the genomic changes that are facilitating the rapid emergence of MRSA ST398 and innovate more refined genotyping techniques to better characterize the reservoirs and transmission routes of human ST398 infection. In the current proposal, we will combine sequencing technologies with in vitro functional assays to assess the phylogenomic structure of ST398 (SA-1) and evaluate the genomic elements enabling human colonization and infection (SA-2, SA-3). We will further develop a set of high-resolution genotyping assays that can be applied in large- scale MRSA ST398 investigations (SA-4). To accomplish our proposed aims, we have assembled a highly qualified multi-national team: Dr. Price (PI; U.S.), an expert in environmental health, bacterial genomics, and translational research is uniquely qualified to lead this study; Dr. Keim (co-I; U.S.), a renowned expert in microbial genetics research will contribute to the genomics portion of this project, including the phylogenomics and assay development; Dr. Tara Smith (co-I; U.S.), a highly experienced American rural health researcher will conduct a longitudinal study of ST398 colonization and infection among U.S. farm workers; Drs. Robert Skov (co-I; Denmark) and Jan Kluytmans (co-I; the Netherlands), two clinician-scientists who have spearheaded the MRSA ST398 surveillance programs in their respective countries, will curate European ST398 isolate collections for the aims of this project. Key innovations of the current study include the application of phylogenomics to elucidate the MRSA ST398 sublineages and resolve the relative contribution of food-animal production on the emergence of this pathogen. Analyzing our unparalleled ST398 collections using a pan-genome approach will maximize our ability to discover genomic adaptations associated with human colonization and infection, as well as livestock-independent community dispersal. Successful completion of this project has the potential to immediately advance our understanding of an important antibiotic-resistant pathogen and facilitate our ability to mitigate its emergence.